PROJECT SUMMARY The purpose of this five-year proposal is to provide an integrative and personalized training program for the applicant to transition into an independent academic position in the cardiovascular sciences. The long-term goal is to discover new treatments for cardiovascular disease through investigating the signaling mechanisms upstream of genome-wide associations. The applicant already has a strong background in cardiovascular pharmacology, physiology, and signal transduction by employing both in vitro and in vivo experimental approaches. This career development plan (K99 phase) will provide additional training in human genetics and genomic analyses employing innovative next-generation sequencing technologies, as well as sophisticated gene-targeting approaches to investigate the regulatory function of causal variants associated with coronary heart disease. The applicant will also receive a wealth of informal and didactic training at Stanford University i specialized areas such as genomic and statistical analyses, and professional development skills, which will be critical for the applicant to gain autonomy and launch a productive career as an independent investigator. Under the expert mentorship of Dr. Thomas Quertermous, MD and the assembled advisory committee (Dr. Assimes, Dr. Tsao, Dr. Pritchard, and Dr. Greenleaf) the applicant will receive the necessary guidance and resources to accomplish these goals and efficiently transition to independence during the R00 phase. The research topic of this proposal fulfills a significant knowledge gap in the field by identifying the missing mechanistic links between common genetic variants and heritable coronary heart disease. As much as half the risk of developing atherosclerotic coronary heart disease (CHD) is genetically predetermined. Genome-wide association studies have now identified multiple independent genetic regions which together account for a fraction (~10%) of the estimated heritability for coronary heart disease (~60%). Gene-gene (epistatic) and gene-environment interactions between common susceptibility regions are predicted to explain this hidden heritability. However, the causal mechanisms by which genetic variation alters disease risk remain largely unknown. Deciphering these regulatory networks is predicted to reveal new insights into disease risk and therapeutic development strategies. Next-generation sequencing approaches have now accelerated our understanding of how non-coding variation alters disease-related gene expression. The goal of this project is to investigate the molecular and genetic interactions responsible for driving risk t two genes, the vascular development transcription factor, TCF21, and the platelet-derived growth factor D (PDGFD). Both of these genes have been associated with heart disease risk in multiple racial/ethnic groups, act independently of traditional risk factors, and are key regulator of smooth muscle cell responses during cardiac development and injury. We previously identified a regulatory mechanism for the TCF21 association involving both transcriptional and post-transcriptional regulation of TCF21 gene expression in coronary artery smooth muscle cells. Recently, we systematically mapped TCF21 transcription factor protein to directly bind and trans-activate the PDGFD locus, and observed a positive correlation of TCF21 and PDGFD expression in human atherosclerotic lesions. Consistently, PDGF-mediated signaling was enriched at TCF21 binding sites and CHD risk loci overall. Based on these findings, we hypothesize that regulation of TCF21 may be pivotal to the SMC phenotypic response to injury in the vessel wall, and that molecular and genetic interactions between the TCF21 and PDGFD loci potentiates expression of these genes, leading to increased coronary atherosclerosis risk. This hypothesis will be empirically tested by pursuing the following specific aims: 1) Dissect the causal mechanisms of regulatory variants at TCF21 and PDGFD loci. 2) Investigate the molecular interactions between the TCF21 and PDGFD loci. To achieve these aims, we will integrate high-throughput genomics in primary diseased human coronary artery tissue with RNA-guided genome targeting in human coronary artery SMC and in mouse embryos in vivo. Ultimately, this work will shed new light on targeting critical pathways associated with hidden CHD risk in various human populations, and enable the development of the next generation of therapeutics.